Hisashi Hatanaka

Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4–ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

Identification of Novel Isoforms of the EML4-ALK Transforming Gene in Non–Small Cell Lung Cancer

The genome of a subset of non-small-cell lung cancers (NSCLC) harbors a small inversion within chromosome 2 that gives rise to a transforming fusion gene, EML4-ALK, which encodes an activated protein tyrosine kinase. Although breakpoints within EML4 have been identified in introns 13 and 20, giving rise to variants 1 and 2, respectively, of EML4-ALK, it has remained unclear whether other isoforms of the fusion gene are present in NSCLC cells. We have now screened NSCLC specimens for other in-frame fusion cDNAs that contain both EML4 and ALK sequences. Two slightly different fusion cDNAs in which exon 6 of EML4 was joined to exon 20 of ALK were each identified in two individuals of the cohort. Whereas one cDNA contained only exons 1 to 6 of EML4 (variant 3a), the other also contained an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). The protein encoded by the latter cDNA thus contained an insertion of 11 amino acids between the EML4 and ALK sequences of that encoded by the former. Both variants 3a and 3b of EML4-ALK exhibited marked transforming activity in vitro as well as oncogenic activity in vivo. A lung cancer cell line expressing endogenous variant 3 of EML4-ALK underwent cell death on exposure to a specific inhibitor of ALK catalytic activity. These data increase the frequency of EML4-ALK-positive NSCLC tumors and bolster the clinical relevance of this oncogenic kinase.

Epigenetic silencing of AXIN2 in colorectal carcinoma with microsatellite instability.

Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44 000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI− CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5′-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.

Chromosome copy number analysis in screening for prognosis-related genomic regions in colorectal carcinoma

Colorectal carcinoma (CRC) remains the major cause of cancer death in humans. Although chromosomal structural anomaly is presumed to play an important role in the carcinogenesis of CRC, chromosomal copy number alterations (CNA) and loss of heterozygosity (LOH) have not yet been analyzed extensively at high resolution in CRC. Here we aim to identify recurrent CNA and LOH in human CRC with the use of single nucleotide polymorphism‐typing microarrays, and to reveal their relevance to clinical outcome. Surgically resected CRC specimens and paired normal mucosa were obtained from a consecutive series of 94 patients with CRC, and both of them were subjected to genotyping with Affymetrix Mapping 50K arrays. CNA and LOH were inferred computationally on every single nucleotide polymorphism site by integrating the array data for paired specimens. Our large dataset reveals recurrent CNA in CRC at chromosomes 7, 8, 13, 18, and 20, and recurrent LOH at chromosomes 1p, 4q, 5q, 8p, 11q, 14q, 15q, 17p, 18, and 22. Frequent uniparental disomy was also identified in chromosomes 8p, 17p, and 18q. Very common CNA and LOH were present at narrow loci of <1 Mbp containing only a few genes. In addition, we revealed a number of novel CNA and LOH that were linked statistically to the prognosis of the patients. The precise and large‐scale measurement of CNA and LOH in the CRC genome is efficient for pinpointing prognosis‐related genome regions as well as providing a list of unknown genes that are likely to be involved in CRC development. (Cancer Sci 2008; 99: 1835–1840)

High-resolution analysis of chromosome copy number alterations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified, with single nucleotide polymorphism-typing microarrays

Angioimmunoblastic T-cell lymphoma (AILT) and peripheral T-cell lymphoma, unspecified (PTCL-u) are relatively frequent subtypes of T- or natural killer cell lymphoma. To characterize the structural anomalies of chromosomes associated with these disorders, we here determined chromosome copy number alterations (CNAs) and loss of heterozygosity (LOH) at >55 000 single nucleotide polymorphism loci for clinical specimens of AILT (n=40) or PTCL-u (n=33). Recurrent copy number gain common to both conditions was detected on chromosomes 8, 9 and 19, whereas common LOH was most frequent for a region of chromosome 2. AILT- or PTCL-u-specific CNAs or LOH were also identified at 21 regions, some spanning only a few hundred base pairs. We also identified prognosis-related CNAs or LOH by several approaches, including Coxs proportional hazard analysis. Among the genes that mapped to such loci, a poor prognosis was linked to overexpression of CARMA1 at 7p22 and of MYCBP2 at 13q22, with both genes being localized within regions of frequent copy number gain. For a frequent LOH region at 2q34, we also identified IKAROS family zinc-finger 2 cDNAs encoding truncated proteins. Our data indicate that AILT and PTCL-u consist of heterogeneous subgroups with distinct transforming genetic alterations.

Risk factors for recurrent bile duct stones after endoscopic papillary balloon dilation: long-term follow-up study.

Background:  Little is known about the long‐term results of endoscopic papillary balloon dilation (EPBD) for bile duct stones.

Diagnostic and Therapeutic Endoscopic Retrograde Cholangiography Using a Short-Type Double-Balloon Endoscope in Patients With Altered Gastrointestinal Anatomy: A Multicenter Prospective Study in Japan

OBJECTIVES:To evaluate the utility and safety of a short-type double-balloon endoscope (DBE) in the treatment of biliary disease in patients with surgically altered gastrointestinal (GI) anatomy.METHODS:This study was conducted as a multicenter, single-arm, prospective trial at five tertiary academic care centers and three community-based hospitals in Japan. Consecutive patients with biliary disease with altered GI anatomy were prospectively included in this study.RESULTS:A total of 311 patients underwent double-balloon endoscopic retrograde cholangiography (ERC). The success rate of reaching the target site, the primary end point, was 97.7% (95% confidence interval (CI): 95.4–99.1). The success rate of biliary cannulation and contrast injection of the targeted duct, the secondary end point, was 96.4% (95% CI: 93.6–98.2), and the therapeutic success rate was 97.9% (95% CI: 95.4–99.2). Adverse events occurred in 33 patients (10.6%, 95% CI: 7.1–14.0) and were managed conservatively in all patients with the exception of 1 in whom a perforation developed, requiring emergency surgery.CONCLUSIONS:ERC using a short-type DBE resulted in an excellent therapeutic success rate and a low rate of adverse events. This treatment can be a first-line treatment for biliary disease in patients with surgically altered GI anatomy.

Identification of transforming activity of free fatty acid receptor 2 by retroviral expression screening

Gallbladder cancer (GBC) is a highly fatal malignancy in humans. Genetic alterations in KRAS or TP53 as well as overexpression of ERBB2 have been shown to contribute to the development of certain types of GBC. However, many cases of GBC do not harbor such genetic changes, with other transforming events awaiting discovery. We here tried to identify novel cancer‐promoting genes in GBC, with the use of a retroviral cDNA expression library. A retroviral cDNA expression library was constructed from a surgically resected clinical specimen of GBC, and was used to infect 3T3 fibroblasts in a focus formation assay. cDNA incorporated into the transformed foci was rescued by PCR. One such cDNA was found to encode free fatty acid receptor 2 (FFAR2), a G protein‐coupled receptor for short‐chain fatty acids. The oncogenic potential of FFAR2 was confirmed both in vitro with the focus formation assay and by evaluation of cell growth in soft agar as well as in vivo with a tumorigenicity assay in nude mice. The isolated FFAR2 cDNA had no sequence alterations, suggesting that upregulation of FFAR2 expression may contribute to malignant transformation. Indeed, all of quantitative RT‐PCR, in situ hybridization, and immunohistochemical analyses showed that the amount of FFAR2 mRNA and its protein product was increased in digestive tract cancer specimens. Furthermore, short‐chain fatty acids potentiated the mitogenic action of FFAR2 in 3T3 cells. Our data thus, for the first time, implicate FFAR2 in carcinogenesis of the digestive tract. (Cancer Sci 2009)

MicroRNA expression profiles of human leukemias.

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Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.

Biphenotypic acute leukemia (BAL) is a relatively rare subtype of acute leukemia characterized by the presence of both myeloid and lymphoid cell surface antigens. We have now screened for transforming genes in BAL blasts with the use of the focus formation assay with a retroviral cDNA expression library constructed from malignant blasts isolated from a BAL patient. Some of the retroviral inserts recovered from transformed foci were found to encode wild-type purinergic receptor P2Y, G protein coupled, 8 (P2RY8). The oncogenic potential of P2RY8 was confirmed with the in vitro focus formation assay as well as with an in vivo tumorigenicity assay in nude mice. A variety of luciferase-based reporter assays revealed that P2RY8 increased both the trans-activation activities of CREB and Elk-1 as well as the transcriptional activities of the serum response element and enhancer-promoter fragments of the c-Fos and c-Myc genes. Quantitation of P2RY8 mRNA in CD34+ cells of bone marrow showed that P2RY8 expression is frequently increased in leukemia patients, especially in those with refractory disease. Our data thus reveal an abundant expression of P2RY8 in leukemic cells and its unexpected role in the pathogenesis of acute leukemia.